This invention relates to treatment of blood cells, and the use of treated, modified blood cells in connection with certain abnormal mammalian physical conditions and disease states. More specifically, it relates to modified mammalian blood and blood cells, methods of achieving the modified mammalian blood and blood cells, and the treatment, among other disorders, of vascular disorders associated with deficient endothelial function, such as vasospatic disorders, in a mammalian patient by administration to the patient of such modified blood and blood cells.
The control and regulation of blood flow through the cardiovascular system of a mammal is well recognized to be of importance in connection with cardiovascular disorders such as atherosclerosis, peripheral vascular disease and many other circulatory disorders. There is an emerging body of literature which indicates that the endothelium plays a major role in the regulation of blood flow through the cardiovascular system. The endothelium is a cellular structure which lines the blood vessels, communicating with the smooth muscle layer of the blood vessel walls. Contraction of this muscle layer causes the blood vessels to constrict (vasoconstriction), and relaxation of this muscle layer causes the blood vessels to expand (vasodilation). A normally functioning endothelium effectively controls the smooth muscles of the vessel wall, by secreting vasodilators or vasoconstrictors which diffuse or are carried to the muscle fibres to cause the muscle fibres either to relax or to contract. One such vasodilator secreted by the endothelium is commonly referred to as xe2x80x9cendothelium derived relaxing factorxe2x80x9d (EDRF), but has recently been established to be nitric oxide, a form thereof or a closely related compound. In addition to regulating blood flow, nitric oxide is recognized as having many other actions within the body, including neurotransmis-sion, a smooth muscle controlling function within the gastrointestinal tract, both natural and drug induced analgesia, a role in impotence and in tumour toxicity.
It is believed that defective functioning of the endothelium of a patient is an underlying factor in many cardiovascular diseases observed in mammalian patients. For example, a patient with atherosclerosis has excessive quantities of lipid underlying the endothelium, including oxidized low density lipoprotein (containing cholesterol) which is believed to interfere with the proper functioning of the endothelial cells.
The present invention is based upon the discovery that one can extracorporeally subject an aliquot of a mammalian patient""s blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to certain stressors and create in the aliquot modified leukocytes. On re-introduction of the aliquot to the patient""s body by various routes including intra-muscular injection, the modified leukocytes have certain beneficial effects. One of these effects is stimulation of the activity of a functionally deficient endothelium.
The modified leukocytes of the present invention can be obtained by subjecting the aliquot of the patient""s blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to stressors selected from heat, ultraviolet radiation and oxidative environments such as treatment with ozone/oxygen mixtures, or any combination of such stressors, simultaneously or sequentially.
Thus, according to the present invention, ex vivo treatment of the blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, promotes the subsequent development of modified leukocytes, which are different from the untreated cells that are found in the peripheral blood of normal individuals and patients before receiving this treatment, in that they cause an n vivo effect.
The effects of the modified leukocytes of the present invention, when re-injected into the mammalian patient""s body, are several in number. Firstly, there is an effect on other leukocytes or their progenitors which have not been modified by the externally applied stressors, as a result of cell-to-cell communication, a widely recognised phenomenon among cells of the immune system. The result of injecting the blood subjected to stressors outside the body is the upregulation of specific cell surface markers such as HLA-DR and CD25 on other, non-treated leukocytes in the peripheral blood, circulating in the patient. This is indicative of an enhanced immune system. It appears that the treated leukocytes release cytokines (intercellular messenger peptides and proteins), or stimulate leukocytes of the recipient to do so, initiating a cascade phenomenon which affects a number of the quiescent leukocytes in the peripheral blood and causes them to become stimulated. This apparently leads to improved blood flow at sites in the body far removed from the site of injection of the treated leukocytes.
Secondly, the stimulated leukocytes present in the blood circulation, perhaps through the intermediary of the same or similar cytokines and probably aster physical contact or binding to the endothelium via cell adhesion molecules, act upon the endothelium, either directly or indirectly, to increase the endothelial vasodilator function probably by increasing the production and/or action of vasodilators such as nitric oxide, prostacyclin and/or by inhibiting the production and/or action of vasoconstrictors, so as to increase blood flow. This can be manifested either as a restoration of the function of a portion of the endothelium which has become defective, the portion being close to or remote from the site of injection. Such restoration of function may occur through repair of deficient cells or an enhanced rate of replacement of damaged cells. It can also be manifested as an overall improvement in endothelial function. This increased blood flow resulting from increased endothelial vasodilator function, and the consequent increase in oxygenation of tissues, is indicative of use of the process of the present invention in treatment of patients with vascular disease including those with advanced peripheral vascular disease, those with chronic varicose ulcers, and those at risk of developing gangrene which frequently results in amputation. In general, the leukocytes according to the present invention are indicated for use in treating any type of vascular disease either involving partial or complete blood vessel occlusion leading to restricted blood flow or dysfunction of the mechanisms required to permit adequate vasodilation such that tissues including those of the central nervous system, heart, lungs, gastrointestinal tract, liver, kidneys, placenta or extremities would be acutely or chronically affected in terms of structure or function.